Alcohol extract of dehulled adlay seeds for treating breast cancer

ABSTRACT

The present invention provides a method for treating breast cancer in a subject, which comprises administering to said subject an effective amount of an alcohol extract of dehulled adlay seeds. Preferably, an ethyl acetate sub-fraction of the alcohol extract of dehulled adlay seeds has a better effect in treating breast cancer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to uses of an extract of dehulled adlay seeds;more particularly, to the treatment of breast cancer.

2. Description of the Related Art

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) is a grass crop andmainly planted in India, Taiwan, Japan, and China (Huang, S. L.; Chen,Y. F.; Chiang, W. Amino acids, fatty acids, and proximate composition ofthe seed of adlay. Food Sci. 1994, 21, 67-74). The seeds of adlay arealso known as Chinese pearl barley and soft-shelled Job's tears. Adlayseeds have long been used in traditional Chinese medicine (TCM) to treatinflammation, dysfunctions of the endocrine system, warts, chapped skin,rheumatism, and neuralgia and also as a nourishing food (Li, S. C.Pen-t'sao kang mu (Systematic Pharmacopoeia); China, 1596). Dehulledadlay (DA) is believed to be beneficial to humans, and many processedproducts from adlay are manufactured as healthy foods or foodsupplements (Huang, S. L.; Chen, Y. F.; Chiang, W. Amino acids, fattyacids, and proximate composition of the seed of adlay. Food Sci. 1994,21, 67-74). The DA is composed of about 8% bran and 92% endosperm, andprevious studies showed that the adlay bran (AB) possessesanti-inflammatory, antiproliferative, and antiallergic activities betterthan the endosperm (Lee, M. Y.; Tsai, S. H.; Kuo, Y. H.; Chiang, W.Anti-tumor and anti-inflammatory activity of the methanol extracts fromadlay bran. Food Sci. Biotechnol. 2008, 17, 1265-1271; Lee, M. Y.; Lin,H. Y.; Cheng, F.; Chiang, W. C.; Kuo, Y. H. Isolation andcharacterization of new lactam compounds that inhibit lung and coloncancer cells from adlay (Coix lachryma-jobi L. var. mayuen Stapf) bran.Food Chem. Toxicol. 2008, 46, 1933-1999; Chen, H. J.; Shih, C. K.; Hsu,H. Y.; Chiang, W. Mast celldependent allergic responses are inhibited byethanolic extract of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.)testa. J. Agric. Food Chem. 2010, 58, 2596-2601).

In line with the medicinal uses of adlay, many studies have beenperformed to evaluate its effects. A series of studies onantitumorrelated activities of adlay seeds have been reported.Coixenolide (Tanimura, A. Studies on the anti-tumor component in theseeds of Coix lachryma-jobi L. var. ma-yuen (Roman.) Stapf. II. Thestructure of coixenolide. Chem. Pharm. Bull. 1961, 9, 47-53), fattyacids (Tokuda, H.; Matsumoto, T.; Konoshima, T.; Kozuka, M.; Nishino,H.; Iwashima, A. Inhibitory effects on Epstein-Barr virus activation andanti-tumor promoting activities of Coix seed. Planta Med. 1990, 56,653-654), and a neutral lipid extract (Woo, J. H.; Li, D.; Wilsbach, K.;Orita, H.; Coulter, J.; Tully, E.; Kwin, T. K.; Xu, S.; Gabrielson, E.Coix seed extract, a commonly used treatment for cancer in China,inhibits NFκB and protein kinase C signaling. Cancer Biol. Ther. 2007,6, 2005-2010; Yu, F.; Gao, J.; Zeng, Y.; Liu, C. X. Inhibition of Coixseed extract on fatty acid synthase, a novel target for anticanceractivity. J. Ethnopharmacol. 2008, 119, 252-258; Lu, Y.; Wu, L. Q.;Dong, Q.; Li, C. S. Experimental study on the effect of Kang-Lai-Teinduced apoptosis of human heptoma carcinoma cell HepG2. HepatobiliaryPancreatic Dis. Int. 2009, 8, 267-272) have been claimed as its activecomponents. Lee et al. reported that a methanolic extract of AB hadantitumor and anti-inflammatory activities (Lee, M. Y.; Tsai, S. H.;Kuo, Y. H.; Chiang, W. Anti-tumor and anti-inflammatory activity of themethanol extracts from adlay bran. Food Sci. Biotechnol. 2008, 17,1265-1271) and that cytotoxic lactams have been isolated (Lee, M. Y.;Lin, H. Y.; Cheng, F.; Chiang, W. C.; Kuo, Y. H. Isolation andcharacterization of new lactam compounds that inhibit lung and coloncancer cells from adlay (Coix lachryma-jobi L. var. mayuen Stapf) bran.Food Chem. Toxicol. 2008, 46, 1933-1999). Additionally, the ethylacetate (EA) fraction of AB ethanolic extract (ABE-EA) is shown tosuppress aberrant crypt foci (ACF) in dimethyl hydrazine (DMH)-inducedcolon carcinogenesis (Chung, C. P.; Hsu, H. Y.; Huang, D. W.; Hsu, H.H.; Lin, J. T.; Shih, C. K.; Chiang, W. Ethyl acetate fraction of adlaybran ethanolic extract inhibits oncogene expression and suppressesDMH-induced preneoplastic lesions of the colon in F344 rats through ananti-inflammatory pathway. J. Agric. Food Chem. 2010, 58, 7616-7623).

The modulatory effects in the endocrine system of adlay seeds have beenreported (Hsia, S. M.; Yeh, C.; Kuo, Y. H.; Wang, P. S.; Chiang, W.Effects of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) hullextracts on the secretion of progesterone and estradiol in vivo and invitro. Exp. Biol. Med. 2007, 232, 1181-1194; Hsia, S. M.; Tseng, Y. W.;Wang, S. W.; Kuo, Y. H.; Huang, D. W.; Wang, P. S.; Chiang, W. Effect ofadlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) hull extracts ontestosterone release from rat Leydig cells. Phytother. Res. 2009, 23,687-695). Human breast cancer cells that express estrogen receptor (ER)and prostate cancer cells that express androgen receptor (AR) areclosely involved with cells that secrete hormone; they are regarded asbenign but possibly may turn into a malignant tumor (Ho, C. Y.; Kim, C,F.; Leung, K. N.; Fung, K. P.; Tse, T. F.; Chan, H.; Lau, C. B.Differential anti-tumor activity of Coriolus versicolor (Yunzhi) extractthrough p53- and/or Bcl-2-dependent apoptotic pathway in human breastcancer cells. Cancer Biol. Ther. 2005, 4, e11-e17). Nonetheless, theeffects of AB on endocrine related cancer cells remain unclear.

SUMMARY OF THE INVENTION

In the present invention, the antiproliferative activities against thebreast cancer cell line of extracts from different parts of dehulledadlay are elucidated.

The invention provides a method for treating breast cancer in a subject,which comprises administering to said subject an effective amount of analcohol extract of dehulled adlay seeds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the GC-MS spectrogram of the alcohol extract ofdehulled adlay seeds according to the invention.

FIG. 2 illustrates the HPLC spectrogram of the alcohol extract ofdehulled adlay seeds according to the invention.

FIG. 3 illustrates the effects of ABE and partitioned fractions on thecell proliferation in (A) MCF-7 and (B) MDA-MB-231 cell lines. Cellswere cultured with or without test samples for 24 h and examined usingthe MTT assay. The results are expressed as a percentage of living cellscultured in the presence of the test samples relative to a parallelculture that did not receive any treatment. Each bar represents themean±SD (n=3). (*) p<0.05 compared to the control group. (**) p<0.01compared to the control group.

FIG. 4 illustrates the effects of ABE-EA and its subfractions on thecell proliferation in (A) MCF-7, (B) T-47D, (C) MDA-MB-231, and (D)H184B5F5/M10 cells. Cells were cultured with or without test samples for24 h and examined using the MTT assay. The results are expressed as apercentage of living cells cultured in the presence of test samplesrelative to a parallel culture that did not receive any treatment. Eachbar represents the mean±SD (n=3). (*) p<0.05 compared to the controlgroup. (**) p<0.01 compared to the control group.

FIG. 5 illustrates LC-MS analytical spectrum of (A) total negative-ioncurrents and UV chromatograms and (B) total positive-ion currents and UVchromatograms of ABE-EA-LEF.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a method for treating breast cancer in asubject, which comprises administering to said subject an effectiveamount of an alcohol extract of dehulled adlay seeds and optionally apharmaceutically acceptable carrier or excipient.

The present invention can be more readily understood by reference to thefollowing detailed description of various embodiments of the invention,the examples, and the chemical drawings and tables with their relevantdescriptions. It is to be understood that unless otherwise specificallyindicated by the claims, the invention is not limited to specificpreparation methods, carriers or formulations, or to particular modes offormulating the compounds of the invention into products or compositionsintended for topical, oral or parenteral administration, because as oneof ordinary skill in the relevant arts is well aware, such things can,of course, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meaning:

Often, ranges are expressed herein as from “about” one particular valueand/or to “about” another particular value. When such a range isexpressed, an embodiment includes the range from the one particularvalue and/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the word “about,” it will beunderstood that the particular value forms another embodiment. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to and independently of the other endpoint.As used herein the term “about” refers to ±10%.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally comprising an agent” meansthat the agent may or may not exist.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, unless otherwiserequired by context, singular terms shall include the plural and pluralterms shall include the singular.

The term “subject” as used herein denotes any animal, preferably amammal, and more preferably a human. The examples of subjects includehumans, non-human primates, rodents, guinea pigs, rabbits, sheep, pigs,goats, cows, horses, dogs and cats.

The term “effective amount” of an active ingredient as provided hereinmeans a sufficient amount of the ingredient to provide the desiredregulation of a desired function, such as gene expression, proteinfunction, or the induction of a particular type of response. As will bepointed out below, the exact amount required will vary from subject tosubject, depending on the disease state, physical conditions, age, sex,species and weight of the subject, the specific identity and formulationof the composition, etc. Dosage regimens may be adjusted to induce theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. Thus, it isnot possible to specify an exact “effective amount.” However, anappropriate effective amount can be determined by one of ordinary skillin the art using only routine experimentation.

The term “treating” or “treatment” as used herein denotes reversing,alleviating, inhibiting the progress of, or improving the disorder orcondition to which such term applies, or one or more symptoms of suchdisorder or condition.

The term “carrier” or “excipient” as used herein refers to anysubstance, not itself a therapeutic agent, used as a carrier and/ordiluent and/or adjuvant, or vehicle for delivery of a therapeutic agentto a subject or added to a formulation to improve its handling orstorage properties or to permit or facilitate formation of a dose unitof the composition into a discrete article such as a capsule or tabletsuitable for oral administration. Suitable carriers or excipients arewell known to persons of ordinary skill in the art of manufacturingpharmaceutical formulations or food products. Carriers or excipients caninclude, by way of illustration and not limitation, buffers, diluents,disintegrants, binding agents, adhesives, wetting agents, polymers,lubricants, glidants, substances added to mask or counteract adisagreeable taste or odor, flavors, dyes, fragrances, and substancesadded to improve appearance of the composition. Acceptable carriers orexcipients include citrate buffer, phosphate buffer, acetate buffer,bicarbonate buffer, stearic acid, magnesium stearate, magnesium oxide,sodium and calcium salts of phosphoric and sulfuric acids, magnesiumcarbonate, talc, gelatin, acacia gum, sodium alginate, pectin, dextrin,mannitol, sorbitol, lactose, sucrose, starches, gelatin, cellulosicmaterials (such as cellulose esters of alkanoic acids and cellulosealkyl esters), low melting wax cocoa butter, amino acids, urea,alcohols, ascorbic acid, phospholipids, proteins (for example, serumalbumin), ethylenediamine tetraacetic acid (EDTA), dimethyl sulfoxide(DMSO), sodium chloride or other salts, liposomes, mannitol, sorbitol,glycerol or powder, polymers (such as polyvinyl-pyrrolidone, polyvinylalcohol, and polyethylene glycols), and other pharmaceuticallyacceptable materials. The carrier should not destroy the pharmacologicalactivity of the therapeutic agent and should be non-toxic whenadministered in doses sufficient to deliver a therapeutic amount of theagent.

Preferably, the alcohol extract of dehulled adlay seeds is comprised ina composition. The composition of the invention is preferably a foodcomposition or a pharmaceutical composition.

The alcohol extract of dehulled adlay seeds can be added to aconventional food composition (i.e. the edible food or drink orprecursors thereof) in the manufacturing process of the foodcomposition. Almost all food compositions can be supplemented with thealcohol extract of dehulled adlay seeds of the invention. The foodcompositions that can be supplemented with the alcohol extract ofdehulled adlay seeds of the invention include, but are not limited to,candies, baked goods, ice creams, dairy products, sweet and flavorsnacks, snack bars, meal replacement products, fast foods, soups,pastas, noodles, canned foods, frozen foods, dried foods, refrigeratedfoods, oils and fats, baby foods, or soft foods painted on breads, ormixtures thereof.

The pharmaceutical composition of the invention is preferablyadministered topically or systemically by any method known in the art,including, but not limited to, intramuscular, intradermal, intravenous,subcutaneous, intraperitoneal, intranasal, oral, mucosal or externalroutes. The appropriate route, formulation and administration schedulecan be determined by those skilled in the art. In the present invention,the pharmaceutical composition can be formulated in various ways,according to the corresponding route of administration, such as a liquidsolution, a suspension, an emulsion, a syrup, a tablet, a pill, acapsule, a sustained release formulation, a powder, a granule, anampoule, an injection, an infusion, a kit, an ointment, a lotion, aliniment, a cream or a combination thereof. If necessary, it may besterilized or mixed with any pharmaceutically acceptable carrier orexcipient, many of which are known to one of ordinary skill in the art;see paragraph [0021] for example.

In one preferred embodiment of the invention, the alcohol extract ofdehulled adlay seeds is subjected to a Gas Chromatography-MassSpectrophotometry (GC-MS) assay. The gas chromatography is conductedwith Varian® 450-GC; and the mass spectrophotometry is conducted withVarian® 240-MS; the column is Varian® VF-5 ms 30 m×0.25 mm (I.D. 0.25μm). The temperature program is 150° C. for 5 min; heating to 200° C. ata rate of 10° C./min for 20 min; and heating to 280° C. at a rate of 10°C./min for 25 min. As shown in FIG. 1, the spectrogram obtainedcomprises peaks at retention time of about 13.581 min, about 19.237 min,19.334 min, about 19.435 min, about 19.799 min, about 37.75 min, about38.051 min, about 48.504 min and about 48.819 min (Table 1).

TABLE 1 Retention time (min) Area (%) 13.581 2.65 19.237 35.17 19.3346.68 19.435 20.60 19.799 4.11 37.75 7.70 38.051 11.94 48.504 8.56 48.8192.60

In one preferred embodiment of the invention, the alcohol extract ofdehulled adlay seeds is subjected to a high performance liquidchromatography assay. The column is Reverse phase C18 column (250×4.6 mmi.d.; YMC Co., INC). The column temperature is 40° C. The chromatogramsare extracted at 280 nm and 320 nm. The mobile phase uses Solution A: 5%acetic acid in water; Solution B: 0.5% acetic acid in water/100%acetonitrile (1:1, v/v). The gradient elution program is shown in Table2. As shown in FIG. 2, the spectrogram obtained comprises peaks atretention time of about 7.5 min, about 12.1 min, 14.4 min, about 16.1min, about 16.8 min, about 18.9 min, about 23.3 min, and about 29.1 min.

TABLE 2 Time Flow rate Mobile Mobile (min) (mL/min) phase A (%) phase B(%) 0 1.0 90 10 10 1.0 85 15 20 1.0 84 16 35 1.0 83 17 50 1.0 79 21 551.0 79 21

The dehulled adlay seeds comprise bran and endosperm, therefore, thealcohol extract of dehulled adlay seeds according to the inventionpreferably comprises an alcohol extract of adlay bran and an alcoholextract of adlay endosperm. The manner for obtaining the bran andendosperm from the dehulled adlay seeds is well-known to artisansskilled in this field.

In one preferred embodiment of the invention, the alcohol extract ofdehulled adlay seeds is an ethanol extract of dehulled adlay seeds.

In one preferred embodiment of the invention, the method comprisesadministering to said subject an effective amount of an ethyl acetatesub-fraction of the alcohol extract of dehulled adlay seeds.

In one preferred embodiment of the invention, the method comprisesadministering to said subject an effective amount of an ethyl acetatesub-fraction of the alcohol extract of adlay bran.

In one preferred embodiment of the invention, the method comprisesadministering to said subject an effective amount of an n-hexanesub-sub-fraction of an ethyl acetate sub-fraction of the alcohol extractof adlay bran.

In one preferred embodiment of the invention, the method comprisesadministering to said subject an effective amount of a 1-butanolsub-fraction of the alcohol extract of adlay bran.

In one preferred embodiment of the invention, the method comprisesadministering to said subject an effective amount of an ethyl acetatesub-fraction of the alcohol extract of adlay endosperm.

In one preferred embodiment of the invention, the alcohol extract ofdehulled adlay seeds is prepared according to a process comprising:

-   -   (a) providing dehulled adlay seeds;    -   (b) cutting the dehulled adlay seeds into small pieces; and    -   (c) extracting the small pieces in step (b) with the alcohol to        obtain an extract.

The term “dehulled adlay seeds” as used herein refers to seeds of adlaywithout hulls, testas, coverings, shells, or pods. The manner ofremoving the hulls, coverings, shells or pods from the adlay seeds iswell-known to artisans skilled in this field. If needed, the bran and/orendosperm is further isolated from the dehulled adlay seeds.

The adlay seeds referred to in this invention are not particularlylimited. Preferably, the adlay belongs to Gramineae family, Panicoideaesub-family, and Coix species, or Poales order, Poaceae family, and Coixspecies. More preferably, the adlay is Coix lachryma-jobi, Coixlachryma-jobi L., Coix lachryma-jobi L. var. ma-yuen Stapf, Coixagrestis Lour., Coix arundinacea Lam., Coix exaltata Jacq., Coix lacrymaL.

According to the process of the invention, prior to step (b), thedehulled adlay seeds are preferably dried.

In one preferred embodiment of the invention, step (b) further comprisesblending the small pieces into powder. The manner of cutting and/orblending is well-known to artisans skilled in this field.

The term “an alcohol extract of dehulled adlay seeds” as used hereinrefers to an extract of dehulled adlay seeds obtained by extracting theseeds with an alcohol solution. The manner of extracting the seeds witha solution is well-known to artisans skilled in this field. In onepreferred embodiment of the invention, the dehulled adlay seeds aresoaked in an alcohol solution for extraction.

The ratio (w/v) of the dehulled adlay seeds and the alcohol solution isnot specifically restricted, and can be about 1:1 to about 1:10;preferably about 1:3 to about 1:8; and most preferably about 1:5.

In one preferred embodiment of the invention, the alcohol is selectedfrom the group consisting of methanol, ethanol, n-propanol, isopropanol,n-butanol, iso-butanol, sec-butanol, tert-butanol, and ethanol acetate.More preferably, the alcohol is methanol or ethanol; most preferably,the alcohol is ethanol. The alcohol solution preferably about 90% toabout 99.9% ethanol.

Preferably, the process further comprises (d) obtaining a liquidfraction from the extract, and a solid fraction is removed. The mannerof removing the solid fraction to obtain the liquid fraction iswell-known to artisans skilled in this field.

In one preferred embodiment of the invention, an ethyl acetate or1-butanol sub-fraction of the alcohol extract of dehulled adlay seeds isprepared by further extracting the alcohol extract of dehulled adlayseeds. In one preferred embodiment of the invention, the process forpreparing the alcohol extract of dehulled adlay seeds further comprises(e) extracting the liquid fraction with ethyl acetate or 1-butanol.

The alcohol extract of dehulled adlay seeds and preferably the ethylacetate sub-fraction of the alcohol extract of dehulled adlay seeds areeffective on treating breast cancer in a subject. More preferably, thebreast cancer is an estrogen receptor positive cancer. In anotheraspect, the breast cancer has a wild-type p53 protein.

The following examples are given for the purpose of illustration onlyand are not intended to limit the scope of the present invention.

Example Materials and Methods

General Experimental Procedures. The MCF-7, T-47D, MDAMB-231, andH184B5F5/M10 cell lines were obtained from the Bioresource Collectionand Research Center (Hsinchu, Taiwan). Fetal bovine serum (FBS), minimumessential medium (MEM), MEM-R, Leibovitz's L-15, and RPMI-1640 mediumwere purchased from GIBCO (Grand Island, N.Y.). Penicillin,streptomycin, glutamine,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT),and other chemicals were obtained from Sigma (St. Louis, Mo.). Silicagel (230-400 mesh, Merck, Darmstadt, Germany), Sephadex LH-20 (AmershamBiosciences, Uppsala, Sweden), and a semi-preparative Si column(LiChrosorb Si-60, Merck) were used for column chromatography. Dimethylsulfoxide (DMSO) and other solvents (analytical grade) were purchasedfrom Merck. Infrared (IR) spectra were recorded on a Nicolet Avatar 320FTIR spectrophotometer (Thermo Electron, Akron, Ohio). Optical rotationwas measured on a Jasco P-2000 polarimeter (Hachioji, Tokyo, Japan).Nuclear magnetic resonance (NMR) spectra were run in CDCl₃ or CD₃OD on aVarian unity INOVA-500 or VNMRS 600 (Varian, Palo Alto, Calif.).Electrospray ionization (ESI)-mass spectra (MS) were recorded on aFinnigan MAT LCQ ion-trap mass spectrometer system (Thermoquest, SanJose, Calif.).

Extraction and Fractionation. Adlay seeds (Taichung Shuenyu No. 4) werepurchased from Taichung, Taiwan, 2009. The dried seeds were divided intothe hull, testa, bran, and endosperm by a grinder and gently blown withan electric fan. A total of 25 kg of AB powder was soaked in 125 L of95% ethanol at room temperature for 48 h. The ethanolic extract wascombined and concentrated under reduced pressure at 50° C. to give 2834g of ABE (10.9% of AB). The ABE was suspended in H₂O, which was followedby sequential partitioning with n-hexane (Hex), ethyl acetate (EA), and1-butanol (BuOH) to give Hex-, EA-, BuOH- (ABE-BuOH, 42 g), andwater-soluble fractions (ABE-H₂O, 258 g) according to the reportedliterature (Lee, M. Y.; Tsai, S. H.; Kuo, Y. H.; Chiang, W. Anti-tumorand anti-inflammatory activity of the methanol extracts from adlay bran.Food Sci. Biotechnol. 2008, 17, 1265-1271). The ABE-Hex was thenrepartitioned with 70% ethanol/H₂O to extract the higher polarcomponents in this fraction, and the 70% ethanol/H₂O fraction wascombined with ABE-EA to give 1720 g of ABE-Hex (60.7% of ABE) and 204 gof ABE-EA (7.2% of ABE). The four fractions were screened forantiproliferative potency on MCF-7 and MDA-MB-231 cells.

Cell Lines and Culture. MCF-7, T-47D, and H184B5F5/M10 cells werecultured with 5% CO₂ atmosphere at 37° C.; MDA-MB-231 cells werecultured without CO₂ at 37° C. The MCF-7 cells were cultured in MEMcontaining 10% heat-inactivated FBS, 100 units/mL penicillin, 100 μg/mLstreptomycin, and 1 mM sodium pyruvate. The T-47D cells were maintainedin RPMI-1640 medium containing 10% heat inactivated FBS, 100 units/mLpenicillin, and 100 μg/mL streptomycin. The H184B5F5/M10 cells weregrown in MEM-R medium containing 10% heat-inactivated FBS, 100 units/mLpenicillin, and 100 μg/mL streptomycin as previously described (Ho, C.Y.; Kim, C, F.; Leung, K. N.; Fung, K. P.; Tse, T. F.; Chan, H.; Lau, C.B. Differential anti-tumor activity of Coriolus versicolor (Yunzhi)extract through p53- and/or Bcl-2-dependent apoptotic pathway in humanbreast cancer cells. Cancer Biol. Ther. 2005, 4, e11-e17). TheMDA-MB-231 cells were cultured in Leibovitz's L-15 containing 10%heat-inactivated FBS, 100 units/mL penicillin, and 100 μg/mLstreptomycin (Woo, J. H.; Li, D.; Wilsbach, K.; Orita, H.; Coulter, J.;Tully, E.; Kwin, T. K.; Xu, S.; Gabrielson, E. Coix seed extract, acommonly used treatment for cancer in China, inhibits NFκB and proteinkinase C signaling. Cancer Biol. Ther. 2007, 6, 2005-2010).

Assay for Antiproliferation. To determine the antiproliferative effect,MCF-7, T-47D, MDA-MB-231, or H184B5F5/M10 cells were assessed using theMTT assay as previously reported (Ho, C. Y.; Kim, C, F.; Leung, K. N.;Fung, K. P.; Tse, T. F.; Chan, H.; Lau, C. B. Differential anti-tumoractivity of Coriolus versicolor (Yunzhi) extract through p53- and/orBcl-2-dependent apoptotic pathway in human breast cancer cells. CancerBiol. Ther. 2005, 4, e11-e17). All of the test samples mentioned abovewere dissolved in DMSO, and the final concentration of DMSO was ≦0.1%.In addition, 0.1% DMSO was used as a blank vehicle. MCF-7, T-47D,MDA-MB-231, or H184B5F5/M10 cells were cultured in 96-well plates at adensity of 1×10⁵ cells/well and allowed to grow for 18-24 h. After this,the medium was replaced by FBS-free MEM, RPMI-1640, L-15, or MEM-R; thenthe various test samples in DMSO were added, and the cells wereincubated for an additional 24 h. Next, MTT solution in FBS-free MEM,RPMI-1640, L-15, or MEMR, which had been filtered through a 0.45 mmmembrane, was added to each well (final concentration of 0.5 mg/mL). Theplates were incubated under a 5% CO₂ atmosphere at 37° C. for 20 min.The medium with unreacted dye was removed. DMSO was added into each wellto dissolve the MTT formazan crystals, and the samples were incubated atroom temperature for 5-10 min. The absorbance at 570 nm was measured,and the viability of cells was calculated using the following equation:relative cell number (%)=(average absorbance of treatedwells)×100/(average absorbance of blank vehicle wells).

Isolation and Purification Process. The total ABE-EA fraction wassubjected to silica gel column chromatography and elution with aHex/EA/MeOH gradient solvent system to give five subfractions, namely,low polarity waste (20% EA/Hex eluate), ABE-EA-A (40% EA/Hex eluate),ABE-EA-B (70% EA/Hex eluate), ABE-EA-C (100% EA eluate), and ABE-EA-D(100% MeOH eluate).

LC-MS Analysis. HPLC analysis was performed using a Finnign MAT pump(P4000, Finnign) with an ultraviolet (UV) detector (UV2000, Finnign).Gradient elution was performed with 0.01% formic acid aqueous solution(v/v, pH 3.30, A) and 50% acetonitrile (ACN) in methanol (v/v, B) at aconstant rate of 0.3 mL/min through an ODS-3 (250×4.6 mm, 5 μm)reverse-phase column (Inertsil). Initial starting conditions were 20% B,at 0-10 min; increase from 20 to 40% B, at 10-30 min; increase from 40to 60% B, at 30-40 min; increase from 60 to 75% B, at 40-50 min;increase from 75 to 78% B, at 50-60 min; increase from 78 to 80% B, at60-70 min; increase from 80 to 100% B, at 70-80 min; and finally,decrease from 100 to 20% B, at original conditions. The absorbance wasset at 280 nm. This system was coupled with a Finnigan MAT LCQ ion-trapmass spectrometer system (Finnigan MAT, San Jose, Calif.), which wasoperated in the ESI mode. An aliquot of the bioactive fraction (10mg/mL, 20 μL) was directly introduced into the column through theautosampler (Finnigan MAT AS3000), with nitrogen being used as thenebulizing and drying gas. The operating parameters used were asfollows: a gas temperature of 250° C., a spray needle voltage of 5 kV, anebulizer pressure of 60 psi, and an auxiliary gas pressure of 30 psi.An ion trap containing helium damping gas was introduced following theprotocols of manufacturer. The mass spectra were acquired in a m/z rangeof 100-1000 with 5 microscans and a maximum ion injection time of 200min. The isolated compounds were confirmed with a retention time (tR)and were qualified by MS using either a negative or positive mode.

Statistical Analysis. The bar values are present as the mean (standarddeviation (SD) from three independent experiments. The results amonggroups were analyzed by analysis of variance (ANOVA) and Student's ttest. A p value of <0.05 is considered to show a significant difference.

Results and Discussion

Effects of ABE and the Various Separated Fractions on the Growth ofHuman Breast Cancer Cells. In the example, the antiproliferation of ABon MCF-7 cells, which is an ER-positive metastatic adenocarcinoma cellline with wild-type p53 protein, on T-47D cells, which is an ER-positiveductal carcinoma but with a mutant p53 protein, and on MDAMB-231 cells,which is an ER-negative carcinoma, involved evaluation of theantiproliferative effects of various subfractions and isolated compoundsfrom ABE. In these experiments, H184B5F5/M10 cells are normal humanbreast epithelia cells used as a control.

As can be seen in FIG. 3, ABE-EA and ABE-BuOH show significantantiproliferative effects on MCF-7 and MDA-MB-231 cells. ABE-EA showsmore potent activities than other fractions; at the same time, thequantitative proportion of ABE-EA in ABE is much greater than ABE-BuOH.Therefore, ABE-EA was used for further fractionation. SubfractionsABE-EA-A-ABE-EA-D showed significant inhibitory effects on human breastcancer cell lines at concentrations of 25-100 μg/mL compared to thecontrol (FIG. 4). However, the ABE-EA-C and ABE-EA-D also suppressed theviability of H184B5F5/M10 cells at 100 μg/mL. On the basis of the aboveresult, ABE-A and ABE-B (40-70% EA/Hex eluate) were selected to isolatethe antiproliferative components. LCF, Drogendorff's reagent positivesubfraction, was obtained and subjected to further purification.

LC-MS Analysis. LC-MS was applied to analyze ABE-EA. The total ioncurrent (TIC) (lane 1) and UV chromatogram (lane 2) of LC-MS for thebioactive fraction are shown in FIG. 5.

While embodiments of the present invention have been illustrated anddescribed, various modifications and improvements can be made by personsskilled in the art. It is intended that the present invention is notlimited to the particular forms as illustrated, and that all themodifications not departing from the spirit and scope of the presentinvention are within the scope as defined in the following claims.

What is claimed is:
 1. A method for treating breast cancer in a subject,which comprises administering to said subject an effective amount of analcohol extract of dehulled adlay seeds.
 2. The method according toclaim 1, wherein the alcohol extract of dehulled adlay seeds is analcohol extract of adlay bran and an alcohol extract of adlay endosperm.3. The method according to claim 1, wherein the alcohol extract ofdehulled adlay seeds comprises an ethanol extract of adlay bran and analcohol extract of adlay endosperm.
 4. The method according to claim 1,which comprises administering to said subject an effective amount of anethyl acetate sub-fraction of the alcohol extract of dehulled adlayseeds.
 5. The method according to claim 1, which comprises administeringto said subject an effective amount of an ethyl acetate sub-fraction ofthe alcohol extract of adlay bran.
 6. The method according to claim 1,which comprises administering to said subject an effective amount of ann-hexane sub-sub-fraction of an ethyl acetate sub-fraction of thealcohol extract of adlay bran.
 7. The method according to claim 1, whichcomprises administering to said subject an effective amount of a1-butanol extract sub-fraction of the alcohol extract of adlay bran. 8.The method according to claim 1, which comprises administering to saidsubject an effective amount of an ethyl acetate sub-fraction of thealcohol extract of adlay endosperm.
 9. The method according to claim 1,wherein the alcohol extract of dehulled adlay seeds is preparedaccording to a process comprising: (a) providing dehulled adlay seeds;(b) cutting the dehulled adlay seeds into small pieces; and (c)extracting the small pieces in step (b) with the alcohol to obtain anextract.
 10. The method according to claim 1, wherein the adlay is Coixlachryma-jobi L. var. ma-yuen Stapf.
 11. The method according to claim1, wherein the alcohol is selected from the group consisting ofmethanol, ethanol, n-propanol, isopropanol, n-butanol, iso-butanol,sec-butanol, tert-butanol, and ethanol acetate.
 12. The method accordingto claim 1, wherein the alcohol is ethanol.
 13. The method according toclaim 9, wherein step (b) further comprises blending the small piecesinto powder.
 14. The method according to claim 9, wherein the processfurther comprises a step (d) obtaining a liquid fraction from theextract.
 15. The method according to claim 14, wherein the processfurther comprises a step (e) extracting the liquid fraction with ethylacetate.
 16. The method according to claim 14, wherein the processfurther comprises a step (e) extracting the liquid fraction with1-butanol.
 17. The method according to claim 1, wherein the breastcancer is an estrogen receptor positive cancer.
 18. The method accordingto claim 1, wherein the breast cancer has a wild-type p53 protein. 19.The method according to claim 1, wherein the alcohol extract of dehulledadlay seeds is comprised in a composition.
 20. The method according toclaim 19, wherein the composition is a food composition or apharmaceutical composition.